|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1990 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1989 : ¥800,000 (Direct Cost : ¥800,000)
Lysosomal cathepsins are considered to play nd important role in intracellular protein degradation. Like most lysosomal acid hydrolases, cathepsins are synthesized as the glycosylated preproenzymens on membrane-bound ribosomes of endoplasmic reticulum and the higher-molecular-weight precursors are subsequently converted into the mature enzymes posttranslationally by limited proteolysis.
In my studies, I have found the following evidence : (1) The latent proforms of cathepsins B, H, L, and D were identified by immunoblot analysis in the rat liver microsomal lumen, and the in vitro activation experiments with liver microsomal contents revealed that latent procathepsins are converted to the active forms of mature enzymes under acidic conditions. (2) The proteolytic processing events, and the activation of the enzymes were strongly inhibited by pepstatin, therefore, suggesting that cathepsin D, a major lysosomal aspartic proteinase is involved in the propeptide-processing of procathepsins.
(3) To further characterize these intracellular processing and activation mechanisms for the lysosomal cathepsins, microsomal procathepsin B was highly purified by the Con A-Sepharose, Sepharose-Gly-Phe-GlySc, and the anti-cathepsin B IgG Sepharose and then this proenzyme was incubated with rat liver tritosomal contents in the presence of several proteinase inhibitors. The result confirmed that the lysosomal cathepsin D is indeed a processing proteinase of procathepsin B. Furthermore, the processed form of procathepsin after the digestion with cathepsin D was isolated and subjected to the determination of the NH_2-terminal sequence of the molecule. The determined NH_2-terminal sequence of procathepsin B revealed to be Pro-66 which is 14 amino acids upstream of the NH_2-terminus of the mature cathepsin B, suggesting that the procathepsin B would undergo multiple processing step during intracellular transport. (4) The multicopy plasmid containing the cathepsin L cDNA has been introduced into the yeast to determine the intracellular sorting machinery of lysosomal cathepsin L. These in vivo and in vitro studies will help me to clarify the extracellular processing and activation mechanisms lysosomal cathepsins. Less